High-performance micropropagation of dendroenergetic poplar hybrids in photomixotrophic Temporary Immersion Bioreactors (TIBs)

Abstract

By the first time an efficient methodology for micropropagation of poplar hybrids by temporary immersion in liquid medium was developed under photomixotrophic conditions. Influence of factors, such as plant regulator/balance in culture medium, number of explants or inoculums per bottle, immersion frequencies in Temporary Immersion Bioreactors (TIBs), reduced sucrose as priming treatment and photoperiod were studied in TIBs with 0.4MPa CO2 at a light intensity of 100μMm−2s−1. Protocol was standardized in three poplar clones recommended for their dendroenergetic traits. The highest multiplication rates were obtained by inoculating 20 and 24 nodal segments in TIBs with 200ml of medium MS+0.22μM TDZ. During winter (8h photoperiod), the highest multiplication rates in TIBs were obtained with 8h immersion frequencies, while in summer (14h photoperiod) immersions every 6h showed the best multiplication rates. The main percent of ex vitro rooted plants was recovered when plantlets were micropropagated in liquid medium supplemented with 1.5% sucrose. A drying pretreatment for 20min followed by 10min inoculation with a mycorrhiza solution (Scleroderma spp.-Pisolithus tinctorius) allowed us to obtain 97.3% of poplars adapted to ex vitro environmental conditions. DNA patterns amplified with the ISSR primer (CTC)5 showed high genomic similarity without polymorphic bands between samples of the same hybrid, which indicated the non-occurrence of genomic changes in the studied populations. Overall, TIBs technology constitute an advance to the industrial development of clonal propagation of poplars for biomass production, in comparison with the classical micropropagation.